1. Field of the Invention
The present invention relates generally to methods and systems for high throughput screening of ligand-receptor bindings in living cells.
2. Background Art
High-throughput screening (HTS) research has benefited from many technological advances over the past few years. The ability to synthesize huge chemical libraries and the availability of automated robots to assist in the screening process have enabled drug development companies to routinely screen a large number of compounds per day.
HTS in vitro and in vivo assays are essential for compound evaluation. Cell-based assays, especially those using optical endpoints, permit investigation of a wide variety of functional properties of these compounds including specific intracellular biochemical pathways, protein-protein interactions, and subcellular localization of the targets. Integration of combinatorial chemistry and HTS with contemporary pharmacology is an important part of drug discovery and development.
G protein-coupled receptors (GPCRs) are a particularly important class of cell surface receptors. The medical importance of these receptors is evidenced by the fact that more than 60% of all commercially available prescription drugs work by interacting with known GPCRs. Hundreds, if not thousands, of receptors convey messages through heterotrimeric G proteins, of which at least 17 distinct forms have been isolated. Most G protein-coupled receptors are comprised of a single protein chain that is threaded through the plasma membrane seven times. Such receptors are often referred to as seven-transmembrane receptors (STRs). More than a hundred different GPCRs have been found, including many distinct receptors that bind the same ligand, and there are likely many more GPCRs awaiting discovery. The development of new drug discovery assays to identify novel modulators of GPCRs would be of tremendous benefit.
Cell membrane receptor proteins are high value targets for therapeutic use. Examples of membrane receptors include the G-protein coupled receptors (GPCRs) and receptors of tyrosine kinases (RTKs). Membrane receptors may be involved in immune system regulation, autonomic nervous system transmission, inflammatory responses, cell differentiation and proliferation, and physiological senses such as smell and sight.
In many biological processes, ligand-receptor binding (LRB) touches off a cascade of cell signaling events. In addition to these signaling events, a common path for GPCR proteins is the internalization of the ligand-bound receptor in an endocytic vesicle. Endocytosis is also observed for a number of RTKs as well. Because of the importance of GPCRs and RTKs as targets for drug discovery, several methods have been developed to study LRB-induced endocytosis. These methods include patch-clamp measurement of cell membrane capacitance (Cm), immunochemical methods, and the use of green fluorescent protein (GFP) tags.
Membrane capacitance measurements require ion currents, which in the case of GPRCs is seldom viable. Immunochemical methods usually require large numbers of cells for the assay. Furthermore, immunoassays can take tens of minutes. GFP tagging GPCR proteins has been used, but has the following drawbacks: (i) they cannot be applied to wild-type GPCRs; (ii) the GPCR-GFP signal is too weak to monitor single endocytic vesicles; (iii) the GFP tagging may alter some properties of native GCPRs. Similar problems arise with the sensitivity and engineering GFP-tagged RTKs. Because of the importance of GPCRs and RTKs in therapeutic application, there remains a need for methods of sensitive assays that can be used in HTS screening.